Ring-opened purine compounds for labeling polynucleotides

ABSTRACT

The invention provides methods for attaching detectable labels to the N 7  -formyl group, of imidazole ring-opened alkylated purines and purine derivatives, directly, through a linking group, or by subsequent derivatization of a blocking group. The invention provides simple and efficient methods for labeling polynucleotides. In addition, the invention provides derivatized ring-opened alkylated purines and purine derivatives, including reagents for chemical and enzymatic synthesis of detectably labeled polynucleotides.

This is a divisional of Ser. No. 08/241,385, May 3, 1994, now U.S. Pat.No. 5,593,829 which is a continuation of Ser. No. 07/802,815 filed Dec.6, 1991, now abandoned.

The present invention relates to methods for labeling nucleosides andpolynucleotides by purine ring-opening reactions, to labeled nucleosidesand polynucleotides obtained by the ring-opening reactions, and to theuse of the labeling reactions and labeled products to detectpolynucleotides in a sample.

BACKGROUND OF THE INVENTION

The invention described herein was developed, in part, using fundsprovided by the National Institutes of Health, Department of Health andHuman Services, Grant No. 2-R01 DE 04321.

The detection of polynucleotides is important in many areas of molecularbiology, biochemistry, biology, pharmacology and clinical medicine,inter alia. Sometimes it is important to detect the presence per se ofany polynucleotide in a sample, as is the case particularly forproducing protein-based therapeutic agents intended for human use, forinstance. Other times it is desired to detect a specific polynucleotidesequence present in a multitude of sequences. For instance, thediagnosis of genetic disorders depends on sequence-specificpolynucleotide detection and sequence-specific nucleic acidhybridization probes play a central role in molecular biology researchwhere they are used for the detection of specific, complementary nucleicacid sequences present in minute amounts in a background of largequantities of other polynucleotides. Assays involving nucleic acidhybridization in the future will be ubiquitous in the clinical lab, andwill find widespread application in other areas, including, forinstance, veterinary medicine, forensic medicine, plant breeding andother areas of agriculture.

Whether it is desired to detect the presence, per se, of polynucleotidesin sample, or to detect a particular polynucleotide sequence, a key stepis labeling the polynucleotide or polynucleotide probe with a detectablelabel. Generally, the specificity and sensitivity of a detection methodwill be at least partly determined by the properties of the detectablelabel and the efficiency of the labeling method used to introduce thelabel into the polynucleotide.

Various labeling techniques have been developed that provide theselectivity and efficiency necessary to detect DNA present at fairly lowlevels in a sample, and to label hybridization probes sufficiently todetect specific sequences of DNA present in a high background of othersequences. For instance, radioisotopes such as ³ H, ⁴ C, ³² p, ³⁵ S and¹²⁵ I have been incorporated into polynucleotides by metabolic,enzymatic and chemical means to serve as detectable reporter groups toindicate the presence of polynucleotides in a sample. ³² p and ³⁵ S,have proven particularly useful in molecular biology studies in suchtechniques as RNA and DNA sequencing, and the panoply ofblot-hybridization methods used to detect, identify, measure, quantitateand localize specific polynucleotide sequences.

Radioactive labels suffer from several drawbacks, however. First, thereis the risk of human exposure to hazardous levels of radioactivityduring the preparation, use and disposal of reagents containing aradioactive tag. In consequence of the risk associated with humanexposure to radiation there is a need when working with radiation forelaborate and expensive safety precautions.

In addition, the radioisotopes most suitable for use in nucleic acidresearch have relatively short half-lives. For instance, ³² p has ahalf-life of only 14 days, and ³⁵ S has a half-life of only 87 days.Radioactively labeled probes therefore have limited shelf-lives andcannot be prepared and standardized in large batches, well before actualuse. The necessity to prepare probes in small batches close to the timeof actual use incurs economic disadvantages of scale. Furthermore, theinability to prepare and characterize large batches of radioactivelylabeled probes is a barrier to developing polynucleotide baseddiagnostic reagents.

The disadvantages of radioactive labels has led to the development ofalternative techniques for introducing stable non-radioactive,detectable labels into polynucleotides. Thus, methods have beendeveloped to label polynucleotides with, inter alia, biotin, digoxigeninand sulfonate.

Perhaps the most widely used non-radioactive polynucleotide label isbiotin. Biotin binds with exceptional specificity and avidity to theproteins avidin and streptavidin. Thus, these proteins will bind withexceptional efficiency to biotinylated DNA. Both proteins can becross-linked to an enzyme to form an enzymatically active conjugate, inwhich the avidin or streptavidin portion specifically detects the biotinin biotinylated DNA and the enzymatic portion catalyses the formation ofa detectable product, such as a colored or luminescent product, whichcan be determined quantitatively and indicates the presence and theamount of biotinylated DNA in the sample. Avidin-alkaline phosphataseconjugates which are compatible with standard EIA colorimetric reagentshave been widely employed in this type of biotin-based procedure.

Other labels, such as digoxigenin, can be used in analogous fashion.Thus, digoxigenin may be incorporated into the DNA analogously to biotinand the digoxigenin-DNA adduct detected by ELISA or other EIA techniquesusing an enzyme conjugated to an anti-digoxigenin polyclonal ormonoclonal antibody.

In view of the widespread utility of labeled polynucleotides, simple,efficient and reliable methods for labeling polynucleotides are needed.The methods presently available, however, suffer from a number ofdisadvantages. For instance, biotinylated polynucleotides are producedusing biotinylated nucleoside derivatives which must be chemicallysynthesized and then incorporated into a polynucleotide by enzymatic orchemical reactions. Thus, substrates for the preparation of biotinylatedand digoxigeninylated polynucleotides are analogues of the nativenucleoside and deoxynucleoside triphosphates, with chemical groupscovalently coupled to the base. Examples of such analogues are uridineand deoxyuridine triphosphate (UTP and dUTP) coupled via a spacer arm tobiotin (biotin-11-dUTP and biotin-11-UTP), or to digoxigenin(digoxigenin-11-dUTP and digoxigenin-11-UTP), in which the detectablegroup is attached at C5 of the uridine base. See Langer et al., Proc.Nat'l. Acad. Sci., U.S.A. 78: 6633-6637 (1981) and Brigati et al.,Virology 126:36-50 (1983).

Methods for synthesizing biotinylated or digoxigeninylated precursors,however, are time-consuming and require considerable expertise insynthetic chemistry. Moreover, labeling polynucleotides with thesesubstrates generally requires expensive enzymes and exacting reactionconditions. Thus, such substrates cannot be used in many situations andare not suitable for the synthesis of large amounts of labeledpolynucleotide.

Photoreactive derivatives of biotin and digoxigenin, such as thosedescribed by Forster et al., Nucl. Acids Res. 13: 745 (1985), provide anon-enzymatic means of incorporating biotin or digoxigenin intopolynucleotides, including double-stranded DNA (dsDNA), single-strandedDNA (ssDNA) and RNA. The photoreactive derivatives must be chemicallysynthesized, however, a technically demanding and expensive process. Inaddition, the polynucleotides being labeled must be pure since thephotochemical reactions are not specific and contaminating compoundssuch as proteins are readily labeled. The density of labeling is lowwith this procedure, being on the order of one label per 200 baseresidues. Furthermore, polynucleotides labeled using the photoreactivederivatives often bear biotin or digoxigenin on sites involved inWatson-Crick hydrogen bonding, which can deleteriously affect theability of a probe to bind to its target sequence in a sample.

Several chemical procedures for incorporating detectable groups intopolynucleotides have been described. For instance, Stavrianopoulos inU.S. Pat. No. 4,843,122 described a method for biotinylating DNA whereinguanine C8 is activated with 3,4,5-trichloro-aniline and then bonded tobiotin-SH. Reisfeld et al., Biochem. Biophys. Res. Comm. 142:519 (1987),described the bisulfite catalyzed bonding of biotin hydrazide tocytidine N⁴ in DNA. Takahashi et al., Nucleic Acids Res. 17: 4899(1989), described the coupling of biotin aminocaproylhydrazide tosingle-stranded DNA by reaction with glutaraldehyde, possibly resultingin substitution at N⁶ of adenosine, N² of guanosine and N⁴ of cytosine.Sverdlov et al., Biochim. Biophys. Acta. 340:153 (1974), described thesulfonation of cytosine C6, accompanied by substitution of the exocyclicN⁴ amino group with methoxyamine, forming N⁴-methoxy-5,6-dihydro-cytosine-6-sulfonate.

Biotin ligands in polynucleotides labeled according to these proceduresserve as biotin receptor (e.g., avidin and streptavidin) binding sites,allowing detection of biotinylated DNA by ELISA and other EIA methods,inter alia, as described above. Similarly, polynucleotides labeled by N⁴-methoxy-5,6-dihydro-cytosine-6-sulfonate bind a monoclonal antibodyagainst N⁴ -methoxy-5,6-dihydro-cytosine-6-sulfonate and are detected inmuch the same way as biotin by ELISA and other EIA methods.

All of these methods suffer from a variety of disadvantages. Somemethods require cumbersome procedures. Some methods can introduce only asingle molecule of detectable label into each polynucleotide molecule.None of the methods can be used to label polynucleotides in a complexmixture of many different contaminants.

Methods have also been described for incorporating a label into apolynucleotide made by chemical synthesis techniques. For instance,Ruth, DNA 3: 123 (1984), described a method for incorporating detectablelabels into purified polynucleotides of defined sequence by chemicalsynthesis. Ruth described nucleosides bearing functionalized linkerssuitable for use in oligonucleotide synthesizers. Syntheticsingle-stranded oligonucleotides of defined sequence were producedbearing the functionalized linkers on pre-selected bases in thesequence. The functional group could be a detectable label or could bejoined to a detectable label following synthesis. Production of thederivatized nucleosides of Ruth's method requires considerable technicalskill, however, and their utility is limited to labeling syntheticoligonucleotides.

Jablonski et al., Nucleic Acids Res. 14:6115 (1986), showed that thehomobifunctional reagent disuccinimidyl suberate can be used to directlycross-link an oligonucleotide with the enzyme alkaline phosphatase,through a single amine-modified base in the oligonucleotide. Thismethod, however, is limited to oligonucleotides containing theamine-modified base. Furthermore, the alkaline phosphatase may interferewith base pairing by the oligonucleotide in a hybridization assay.

In sum, despite the importance of labeled polynucleotides in molecularbiology, biochemistry and clinical diagnostic applications, inter alia,a simple, reliable, efficient and widely applicable procedure forintroducing detectable labels into polynucleotides has not beendeveloped. All of the procedures discussed hereinabove are of limitedapplicability. Few methods provide for introducing many labels into eachpolynucleotide. Conventional enzymatic labeling methods requireexpensive enzymes, highly purified nucleic acid templates andsubstrates, and reaction conditions must be stringently controlled.Furthermore, the efficiency of labeling by these methods is difficult todetermine, making it hard to assess usefulness of a labeled productbefore use.

Photochemical and chemical (sulfonation) procedures have a wider rangeof applicability but require cumbersome chemical procedures and alsorequire purified nucleic acid substrates. Although many of the prior artprocedures can be completed within a few hours, they may requireextensive preliminary preparations and the entire process is usuallyvery time consuming. Furthermore, excepting photochemical labeling andthe automated synthesis of labeled oligonucleotides, labeling proceduresyield very small amounts (microgram quantities) of labeled product.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a methodfor labeling polynucleotides which is simple, reliable, efficient andsensitive, and can be applied to labeling pure polynucleotides orpolynucleotides in the presence of contaminants.

It is a further aspect of the invention to provide a simple andefficient method to label polynucleotides for use as probes inhybridization assays, inter alia.

Yet another object of the present invention is to provide a simple,efficient and highly sensitive method to quantitatively detectpolynucleotides in a sample.

It is also an object of the present invention to provide a means forintroducing a detectable label into a nucleoside derivative suitable forlabeling a polynucleotide via chemical synthesis or enzymatic-labelingtechniques.

It is a further object of the present invention to provide nucleosidederivatives which comprise detectable labels or functional groupssuitable for bonding to detectable labels.

It is yet another object of the present invention to providepolynucleotides which comprise detectable labels or functional groupssuitable for bonding to detectable labels.

The invention thus provides a variety of means to derivatize nucleosidiccompounds, including nucleosides, nucleoside derivatives,oligonucleotides and polynucleotides, to introduce therein by means of apurine ring-opening reaction, a reactive moiety suitable for attachingthereto a protecting or blocking group, a linking group, or anotherintermediate group, and, ultimately a substituent comprising adetectable label.

In another aspect, the invention provides novel compounds, including butnot limited to ring-opened substituted purine derivatives, and oligo andpolynucleotides comprising the ring-opened substituted purinederivatives. Thus, for instance, the invention provides ring-openedpurine derivatives suitable for use as precursors in syntheticoligonucleotide synthesizers. And, as another example, the inventionprovides derivatized polynucleotides, either detectably labeled, orderivatized so as to facilitate the introduction of a detectable label,which are suitable for use, inter alia, as hybridization probes.

The invention also provides reagents, procedures and kits for labelingpolynucleotides by means of ring-opened purine derivatives, to detect,identify, measure, quantitate or localize polynucleotides in a sample,in either a sequence-specific or a sequence independent manner, or both.

Thus, the invention relates to methods for labeling nucleosides andpolynucleotides by means of purine ring-opening reactions to produce aformyl group which group is then further derivatized to comprise,ultimately, a detectable label, to the products produced by the methods,and to the uses of the methods and the products in detectingpolynucleotides.

In accomplishing the foregoing objects, there has been provided, inaccordance with another aspect of the present invention a method forlabeling a polynucleotide comprising at least one purine nucleotide,comprising the steps of (a) alkylating a purine ring nitrogen in anucleotide in a polynucleotide to form an N-alkylated purine nucleotide;(b) cleaving a bond in the N-alkylated purine nucleotide to form aring-opened N-alkyl-N-formyl nucleotide; and, (c) chemically bonding adetectable label to the N-formyl group, either directly or .through alinking group.

In a preferred embodiment of this aspect of the invention there has beenprovided a method for labeling a polynucleotide that comprises at leastone quanine nucleotide, comprising the steps of (a) N⁷ -alkylating aguanine nucleotide in the polynucleotide to form an N⁷ -alkylguaninenucleotide; (b) cleaving the C8-N⁹ bond in the N⁷ -alkylguaninenucleotide to form a ring-opened N⁷ -formyl-N⁷ -alkylguanine nucleotide;and, (c) chemically bonding a detectable label to the N⁷ -formyl group,either directly or through a linking group.

In a particularly preferred embodiment of the invention there isprovided a method for labeling polynucleotides comprising at least oneguanine nucleotide comprising steps of (a) mixing dimethylsulfate (DMS)in a neutral buffer with the polynucleotide; (b) incubating the DMS andpolynucleotide-containing solution under conditions suitable for N⁷-methylation of the guanine nucleotide in the polynucleotide to form anN⁷ -methylguanine nucleotide; (c) adding a base to the solution of step(b) in an amount sufficient to cleave the bond between C8 and N⁹ in theN⁷ -methyl guanine nucleotide to form a ring-opened N⁷ -formyl-N⁷-methylguanine nucleotide in a polynucleotide in the sample; and,chemically bonding a detectable label to the N⁷ -formyl group of the N⁷-formyl-N⁷ -methylguanine nucleotide, either directly or through alinking group.

In another aspect of the invention, using the foregoing methods, therehas been provided a method for detecting a polynucleotide comprising atleast one purine nucleotide in a sample, comprising the step ofdetecting the polynucleotide in the sample by determining a detectablelabel chemically bonded to an N-formyl group formed in the purinenucleotide.

In accordance with another aspect of the invention there have beenprovided compounds according to the following formula: ##STR1## wherein,A is --H or --OH; B is --H, --OH, mono-, di- or triphosphate or aderivative thereof, or a protecting group for chemical polynucleotidesynthesis; D is --H, --OH, mono-, di- or triphosphate or a derivativethereof, or a protecting group for chemical polynucleotide synthesis; Eis an alkyl group; F is a bond or linking moiety; and, G is a detectablelabel or a blocking group.

In accordance with another aspect of the invention there have beenprovided polynucleotides comprising a compound according to thefollowing formula: ##STR2## wherein: A is --H or --OH; B is --H, --OH,or mono-, di- or triphosphate or a derivative thereof, or aphosphodiester bond or a derivative thereof; D is --H, --OH or mono-,di- or triphosphate or a derivative thereof, or a phosphodiester bond ora derivative thereof; E is an alkyl group; F is a bond or linkingmoiety, and; G is a detectable label.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those in the art from this detailed description.

GLOSSARY

The term N⁷ -formyl-N⁷ -alkylpurine is used herein to designate certainring-opened compounds of the invention which may also be calledalkylformamidopyrimidine or 2,4,5-triamino-6-hydroxy-N⁵ -methyl-N⁵-formylpyrimidine compounds, inter alia, but herein are referred to asN⁷ -formyl-N⁷ -alkylpurine or ring-opened N⁷ -formyl-N⁷ -alkylpurine,and, in the case of guanine and guanosine, N⁷ -formyl-N⁷ -alkylguanineand N⁷ -formyl-N⁷ -alkylguanosine, or ring-opened N⁷ -formyl-N⁷-alkylguanine and ring-opened N⁷ -formyl-N⁷ -alkylguanosine. Anothername for these alkylguanine deoxynucleoside derivatives is2-amino-5-(formyl-alkylamino)-6-(2'-deoxyribosylamino)-pyrimidin-4-one,which for N⁷ -formyl-N⁷ -methylguanosine is2-amino-5-(formyl-methylamino)-6-(2'-deoxyribosylamino)-pyrimidin-4-one.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing the formation of N⁷ -methyl-N⁷-formyldeoxyguanosine by treating deoxyguanosine with dimethylsulfate(DMS) to form N⁷ -methyldeoxyguanosine, and then with base (OH⁻) to openthe alkylated purine ring to form ring-opened N⁷ -formyl-N⁷-methyldeoxyguanosine.

FIG. 2 is a diagram showing two reaction schemes for chemically bondinga detectable label ("detectable label", R') to a formyl substituent(aldehyde) in, inter alia, ring-opened N⁷ -formyl-N⁷-methyldeoxyguanosine. In the reaction pathway on the left side of thediagram, a hydrazide of a detectable label is employed to form ahydrazone upon reacting with the formyl substituent. In the reactionpathway on the right side of the diagram, an amine of a detectable labelis employed to form a Schiff base with the formyl group, which is thenreduced with cyanoborohydride or BH₄ ⁻.

FIG. 3 is a diagram showing two reaction schemes for biotinylating theN⁷ -formyl substituent in ring-opened N⁷ -formyl-N⁷-methyldeoxyguanosine. In the reaction pathway on the left side of thediagram, the hydrazide of aminocaproyl biotin is employed to form biotinaminocaproyl hydrazone upon reacting with the formyl substituent. In thereaction pathway on the right side of the diagram, diaminocaproyl biotinis employed to form a Schiff base upon reacting with the formyl group,which is then reduced with cyanoborohydride or BH₄ ⁻.

FIG. 4 is a more detailed diagram showing the formation of a biotinaminocaproyl hydrazone derivative of ring-opened N⁷ -formyl-N⁷-methyldeoxyguanosine by reacting biotin aminocaproyl hydrazide with theformyl substituent of ring-opened N⁷ -formyl-N⁷ -methyldeoxyguanosine.

FIG. 5 is a graph showing that untreated remains unlabeled in thepresence of tritiated borohydride, but DNA treated in accordance withthe invention incorporates tritium from the tritiating reagent in directproportion to the extent of reaction with DMS.

FIG. 6 is a graph showing that a competitive enzyme assay according tothe invention provides a linear and sensitive measure of DNA in a samplecontaining 1 to 25 nanograms of DNA. The assay was carried out bymeasuring the ability of DNA biotinylated in accordance with theinvention to inhibit binding of a biotin enzyme conjugate to immobilizedavidin.

FIG. 7 is a graph showing that a competitive enzyme assay of theinvention provides a linear and sensitive measure of DNA in a sample ina range of 1 to 100 picograms of DNA. The assay was carried out asdescribed in FIG. 6.

DETAILED DESCRIPTION OF THE INVENTION

The present invention overcomes many of the problems of labelingpolynucleotides found in the prior art. Particularly, in some aspects,it eliminates the need for complex chemical or enzymatic reactions. Itdoes not require the use of highly pure nucleic acids and is universallyapplicable to any polynucleotide containing one or more purine,preferably guanine residues. Moreover, because the label targetsportions of a probe molecule which are not involved with hydrogenbonding to the target nucleic acid, probes labeled according to themethod of the invention are likely to be unimpaired in the recognitionand binding of target nucleic acids. This feature allows higher densityof labeling, thus enhancing the sensitivity of the invention, and thesimplicity of the reaction and lack of a requirement for enzymes rendersthe method of the present invention less prone to error.

Accordingly, the present invention relates to methods for labeling anddetecting polynucleotides by means of a purine ring-opening reaction.More specifically, the present invention relates to an alkylation andring-opening reaction that cleaves the imidazole ring of a purine andintroduces a formyl group into the opened imidazole ring, and, inparticular, to the derivatization of the formyl group to introducedetectable substituents into the ring-opened purine derivatives.

In accordance with the invention the ring-opening and derivatizationreaction may be carried out on a purine nucleoside or a polynucleotidecontaining a purine nucleotide, particularly guanine nucleoside andnucleoside derivatives and guanine nucleotide-containingpolynucleotides, including ribo- and deoxyribopolynucleotides, which maybe single stranded, double stranded, or triple stranded polynucleotides,inter alia.

In one aspect the invention provides for detecting, identifying,measuring, quantitating, or localizing, inter alia, polynucleotides in abiological sample. This aspect of the invention is equally useful todetect polynucleotides in a sequence-independent manner and to detectspecific polynucleotide sequences.

Thus, the invention provides a highly sensitive method to detect, in asequence-independent manner any RNA or DNA in a biological sample, suchas a protein preparation or any other composition prepared from orexposed to a biological source of RNA or DNA, or in which such RNA orDNA might desirably be detected.

Notably, this aspect of the invention provides for the introduction intoeach polynucleotide in a sample of a multiplicity of detectable labelsby the derivatization of multiple purine, preferably guanine,nucleotides in each polynucleotide. The introduction of multipledetectable moieties into each polynucleotide molecule provides anextremely sensitive means of detecting such polynucleotides.

The invention also provides means for labeling polynucleotides which maybe used for sequence-dependent detection, identification, measurement,quantitation or localization of polynucleotides. For instance, thisaspect of the invention provides for labeling a polynucleotide for useas a hybridization probe. In general, individual RNA and DNA sequencesare most useful for this purpose, but any polynucleotide preparationcapable of identifying by base-pairing a polynucleotide of interest canbe employed as a hybridization probe.

Thus, the invention may be employed to label any polynucleotide as ahybridization probe, including, for instance, syntheticoligonucleotides, restriction fragments, plasmids, cosmids, phage, etc.,individual RNAs or DNAs, crude preparations of RNA or DNA, includingtransfer RNA (tRNA), ribosomal RNA (rRNA), messenger RNA (mRNA) ormitochondrial, epigenomic or genomic DNA, or fragments thereof ormixtures thereof.

In general, polynucleotides to be used as hybridization probes (or,indeed, for other purposes) in accordance with the invention may belabeled by chemical synthesis by incorporating a residue previouslyderivatized in accordance with the invention; by enzymatic means, suchas nick translation, "fill-in", transcription, reverse transcription,and the like, to incorporate a residue previously derivatized accordingto the invention into a polynucleotide; and by derivatizing, inaccordance with the invention, a nucleotide within or at one or theother end of a polynucleotide. A variety of such reactions are known, asdescribed, for instance, in Maniatis, et al., MOLECULAR CLONING, ALABORATORY MANUAL (Cold Spring Harbor Laboratory, 1982) and Sambrook, etal., MOLECULAR CLONING, A LABORATORY MANUAL, Second Edition, Vol. 1-3(Cold Spring Harbor Laboratory, 1989).

The invention combines a series of reliable reactions into a newtechnique for specifically coupling a detectable label to purine,preferably quanine residues in nucleosides, nucleoside derivatives,polynucleotides and polynucleotide derivatives, inter alia.

The initial reaction involves the selective alkylation of a purine ringnitrogen by an alkylating agent. In a preferred embodiment, guanine N⁷is alkylated, selectively. Suitable alkylating agents of the inventioninclude but are not limited to dimethylsulfate (DMS) and diethylsulfate,for instance.

In a highly preferred embodiment of the invention, alkylation is carriedout using DMS. When DMS is used in the invention to alkylate DNA, thereaction is preferably carried out at neutral pH. This reaction issimilar to the first step of the guanosine-specific cleavage in theMaxam-Gilbert sequencing technique, which is described in Maxam et al.,Methods in Enzymology 65:449 (1980), inter alia.

The degree of methylation obtained by this reaction can be controlled byvarying the incubation time, the concentration of DMS, and thetemperature of the reaction, among others. The degree of alkylationdetermines the number of imidazole ring-opened guanosine nucleotides andthus, the number of detectable labels that will be chemically bonded toa polynucleotide in accordance with the invention. This provides a meansto tailor the degree of labeling to particular applications. Notably, inthe case of DMS-mediated guanosine methylation in the G-specificreaction in the Maxam-Gilbert DNA sequencing procedure, the degree ofmethylation can be stringently controlled to provide methylation of onlyone G residue in 10, one in 100 or one in 1,000, inter alia, and thesame holds true in the present invention.

N⁷ purine alkylation generally, and N⁷ guanine alkylation in particular,produces a positively charged intermediate. For instance, N⁷-alkylguanosine derivatives have a positive charge on N⁷. Moreover, theN⁷ substituent strongly affects the lability of the C8-N⁹ bond. The moreelectron-accepting the substituents the more labile the bond. Thus,alkylating agents of the invention can be selected to provide a rate ofimidazole ring-opening suitable to a wide variety of reactionconditions, if desired. See, for instance, Kochetkov et al., ORGANICCHEMISTRY OF NUCLEIC ACIDS, PART B, Chapter 7, Plenum Press 1972.

Preferably, hydroxide is employed to cleave the N⁷ -alkylated purine andresults in an imidazole ring-opening reaction. Base mediated cleavage ofN⁷ -alkylated guanine according to the invention normally cleaves theC8-N⁹ bond, opening the purine imidazole ring, providing a ring-openedN⁷ -formyl-N⁷ -alkyl-substituted compound, and this cleavage will beused herein to illustrate the process.

An alkylation and base-mediated ring-opening reaction are described inMaxam et al., supra, inter alia. The invention in the presentapplication, however, provides for chemically bonding detectable labels,inter alia, to N-formyl groups of the ring-opened compounds and differsgreatly from the subsequent steps of the procedure employed in theMaxam-Gilbert sequencing technique. The object in the sequencingtechnique is controlled lysis of the phosphodiester backbone of DNA,whereas in the present invention the backbone remains intact.Accordingly, in the Maxam-Gilbert procedure, base-mediated cleavage ofthe purine imidazole ring and strand scission by an elimination reactionthat severs a phosphodiester bond are catalyzed with piperidine. In thepresent invention, conditions that would lead to strand scission areavoided. Instead, following base-mediated imidazole ring-opening, theN-formyl substituent is coupled to a detectable label, a functionalizedlinker, or a blocking or protecting group.

A wide variety of reactions by means of which a covalent bond is formedbetween a formyl group and another substituent are useful in the presentinvention for forming a covalent bond between the N-formyl group of aring-opened purine and a desired addition substituent.

In a general sense, these reagents may serve three functions, providing(1) a reactive group for bonding to the formyl group of an N⁷ -formyl-N⁷-alkylpurine, (2) a linking moiety, and (3) a second substituent whichmay be another reactive substituent, a blocking group which willgenerally later be removed to generate a reactive group for furtherderivatization, or a detectable label. It will be appreciated, however,that one, two or all three of these functions may be served by a singlechemical entity.

Reactive substituents for forming a chemical bond with the N⁷ -formylsubstituent of an N⁷ -formyl-N⁷ -alkylpurine include any substituentscapable of forming a bond with the N⁷ -formyl group. Generally, thesubstituents will be those that do not deleteriously modify thealkylpurine as a result of the process of bond formation. A deleteriousmodification in this context will be understood to mean a modificationthat interferes with the particular application for which the modifiedalkylpurine is being prepared. Thus, reagents useful for bonding to theN⁷ -formyl group include those useful in the Wittig reaction, thoseuseful in the aldol condensation, amines, hydrazides, semicarbazides,inter alia. Particularly useful reactive substituents for bonding to theN⁷ -formyl group are amines, hydrazides, semicarbazides.

Reactive groups useful in the invention include but are not limited toany that form the following linkages with the carbon atom of theN-formyl group, ═NNHCH₂ R, ═NNHCOR, ═NHCH₂ R, --NHR, ═CHCOR, ═CRR',--OCH(R)R', wherein R and R' simply indicate other portions of thecompounds which may ultimately be chemically bonded to the formyl groupthrough the indicated linkages. It will be understood that R and R' inthis context include the full range of substituents useful as linkingand blocking group, as well as those useful as detectable labels, as setforth elsewhere herein in greater detail.

Preferred groups will be those bearing primary amine or hydrazinefunctions. Amines form Schiff bases with the formyl group, which can bestabilized by reduction to secondary amines, e.g. with NaBH₃ CN.Hydrazines condense with formyl groups to form relatively stablehydrazones, which normally do not require further stabilization. Theamine or hydrazone functions can be part of linkers that are previouslyor subsequently reacted with other moieties, eventually including thedesired detectable label.

The linking group, when it is present in the reagent usually will be alinear or branched carbon chain which may also comprise N or S or both,and generally will comprise at least four atoms. It will be appreciatedthat the exact chemical composition of a linking group will be dictatedby the particular application. Thus, the linking group will be stable tothe reactions required for bonding to the N⁷ -formyl group, and also toother reactions that may be necessary to a given application, such asremoving a blocking group, attaching a detectable label or the like.

Preferred linking groups include, but are not limited to the monomer,dimer or trimer of ε-amino caproic acid (H₂ N-(CH₂)₅ --COOH), and alkanediamines including 1,4-diaminobutane, 1,5-diaminopentane and1,6-diaminohexane, inter alia. Particularly preferred among the ε-aminocaproic acids and similar chains are those which are soluble in aqueousbuffers.

Following formation of the ring-opened purine, the reaction conditionsare adjusted to accommodate the formation of a bond between the N-formylgroup and the reactive moiety of the substituent. Generally, theconditions will be such as not to unfavorably modify the nucleotidesubstrate in any Way. Additional blocking groups, detectable labels andthe like that can be used in accordance with the invention are set forthbelow.

The present invention also relates to compounds of the followingformula, which may be conveniently produced by the methods of thepresent invention. ##STR3## wherein, A is --H or --OH;

B is --H, --OH, mono-, di- or triphosphate or a derivative thereof, or ablocking group for chemical polynucleotide synthesis, or it is aphosphodiester bond or a derivative thereof;

D is --H, --OH, mono-, di- or triphosphate or a derivative thereof, or ablocking group for chemical polynucleotide synthesis, or it is aphosphodiester bond or a derivative thereof;

E is an alkyl group;

F is a bond or linking moiety, and;

G is a detectable label or a blocking group.

Compounds of the invention therefore include but are not limited to riboand deoxyribonucleosides and their derivatives, including DNA and RNA,wherein A is --H or --OH, respectively. Also within the scope of theinvention are compounds of the above formula which comprise derivativesof --OH compatible with methods for synthesizing RNA.

Compounds of the invention include those wherein B is --H or --OH, or amono-, di- or triphosphate or a derivative thereof. Included, amongothers, are RNA and DNA polynucleotides, wherein B will be aphosphodiester or a derivative of a phosphodiester; nucleosides andderivatives thereof, including nucleoside monophosphates, nucleoside 3'diphosphates, and nucleoside 3' triphosphates; dideoxynucleosides andderivatives thereof.

Also included are compounds wherein B is a blocking (or protecting)group suitable for chemically synthesizing DNA or RNA. (The termsblocking group and protecting group are used interchangeably herein torefer to chemical groups that render a reactive substituent in acompound inert to a chemical process and allow the substituent or aderivative thereof subsequently to be regenerated. Such groups are wellknown in the art and are crucial to chemical methods of oligonucleotideand peptide synthesis, for instance.)

A variety of such 3' blocking groups for oligonucleotide synthesis areknown to the art, as, for instance, described in Gait, M. J.,OLIGONUCLEOTIDE SYNTHESIS, A PRACTICAL APPROACH, (IRL Press, 1984) whichis herein incorporated by reference. One preferred blocking group ofthis type is the β-cyanoethyl N,N-diisopropyl phosphoramidite blockinggroup, which is extensively employed in substrates for automatedoligonucleotide synthesis.

Compounds of the invention include those wherein D is --H or --OH, or amono-, di- or triphosphate or a derivative thereof, or a phosphodiesterbond. Included are RNA and DNA polynucleotides, wherein B will be aphosphodiester or a derivative of a phosphodiester; nucleosides andderivatives thereof, including nucleoside 5' monophosphates, nucleoside5' diphosphates, and nucleoside 5' triphosphates; dideoxynucleosides andderivatives thereof, including especially dideoxynucleosidetriphosphates, among others.

Also included are compounds wherein D is a blocking group suitable forchemically synthesizing DNA or RNA. A variety of 5' blocking groups foroligonucleotide synthesis are described in Gait, M. J., OLIGONUCLEOTIDESYNTHESIS, A PRACTICAL APPROACH, (IRL Press, 1984). A preferred blockinggroup of this type is the dimethoxytrityl blocking group extensivelyemployed in substrates for automated oligonucleotide synthesis.

In compounds of the invention illustrated by the above formula, E is analkyl group, which may be a straight-chain, branched-chain, or cyclicalkyl, including alkyl substituted straight-chain, branched-chain, orcyclic alkyl groups. Preferably E will be a primary alkyl, especially alower alkyl group. Highly preferred alkyl groups include methyl, ethyl,or benzyl alkyl groups, of which the methyl group is very highlypreferred.

F is a covalent bonding function formed between the N⁷ -formyl group ofN⁷ -formyl-N⁷ -alkylpurine and G. Most preferably, F comprises either--CH═N-- or --CH₂ N--.

F may be a direct bond formed by an N⁷ -formyl group and a substituentof a detectable label or it may be one or more linking groups forattaching at some distance a detectable label to the N⁷ -formyl group,or it may be an intermediate group, such as blocking group, capable offurther derivatization to chemically bond a detectable label.

Accordingly, F may be a single substituent or it may be a more complexsubstituent. For instance, F may be any C--, N-- or S-- containing groupthat can serve as a linker group. Preferably, if F is a linking group,it will be a linear or branched carbon chain which may also comprise Nor S or both, comprising at least four atoms.

When F comprises a linking group, it will usually have been derived froma divalent compound, that is a compound that contained two reactivegroups, one that coupled to the N⁷ -formyl group and the other to coupleto a second linking group, a blocking group or to a detectable label.

A variety of homobifunctional and heterobifunctional linking reagentsknown in the art are useful in the present invention. These reagentsprovide a reactive substituent for forming a bond with the formyl groupof a ring-opened N⁷ -formyl-N⁷ -alkylpurine of the invention and areactive substituent for forming a bond to a detectable label, or toanother linking group or to a blocking agent.

Preferred linkers include the monomer, dimer or trimer of ε-aminocaproic acid (H₂ N-(CH₂)₅ --COOH), and alkane diamines including1,4-diaminobutane, 1,5-diaminopentane and 1,6-diaminohexane, inter alia.Particularly preferred among the ε-amino caproic acids and similarcompounds are those which are soluble in aqueous buffers.

Reactive substituents comprised by F for forming a chemical bond withthe N⁷ -formyl substituent of an N⁷ -formyl-N⁷ -alkylpurine include anysubstituents capable of forming a bond with the N⁷ -formyl group.Generally, the substituents will be those that do not deleteriouslymodify the alkylpurine as a result of the process of bond formation. Adeleterious modification in this context will be understood to mean amodification that interferes with the particular application for whichthe modified alkylpurine is being prepared. Thus, reagents useful forbonding to the N⁷ -formyl group include those useful in the Wittigreaction, those useful in the aldol condensation, amines, hydrazides,semicarbazides, inter alia. Particularly useful reactive substituentsfor bonding to the N⁷ -formyl group are amines, hydrazides,semicarbazides.

Reactive groups useful in the invention include but are not limited toany that form the following linkages with the carbon atom of theN-formyl group, ═NNHCH₂ R, ═NNHCOR, ═NHCH₂ R, --NHR, ═CHCOR, ═CRR',--OCH(R)R', wherein R and R' simply indicate other portions of thecompounds which may ultimately be chemically bonded to the formyl groupthrough the indicated linkages. It will be understood that R and R' inthis context include the full range of substituents useful as linkingand blocking group, as well as those useful as detectable labels, as setforth elsewhere herein in greater detail.

Preferred groups will be those bearing primary amine or hydrazinefunctions. Amines form Schiff bases with the formyl group, which can bestabilized by reduction to secondary amines, e.g. with NaBH₃ CN.Hydrazines condense with formyl groups to form relatively stablehydrazones, which normally do not require further stabilization. Theamine or hydrazone functions can be part of linkers that are previouslyor subsequently reacted with other moieties, eventually including thedesired detectable label.

Compounds according to the invention may include a detectable label, G.G may be any chemical group which has a physical or chemicalcharacteristic which can be detected, and any label that can be detectedby a physical or chemical method may be used in the invention.

Detectable labels of the invention may be determined by colorimetry,spectrophotometry, fluorimetry, methods for detecting radioactivity, andother methods well-known to the art.

Detectable labels of the invention include labels characterized bycolor, luminescence, fluorescence, radioactivity, ligand recognition,affinity binding, chemical reactivity and enzymatic activity, amongothers.

Included among the affinity binding labels are ligands of all types,lectin binding moieties, substrates and substrate analogs, co-factors,antigens, immunologically reactive substances and substituents such asepitopes, determinants and haptens and the like, and substances thatcomprise epitopes, determinants and haptens and the like, that bindantibodies, antibody derivatives, single chain antibodies and the like.

Included among the fluorescent compounds are7-amino-4-methylcoumarin-3-acetyl hydrazide, fluorescein, rhodamine;among the lectin ligands are lactose, N-acetylglucosamine.N-acetylgalactosamine or mannose; among the antigen or antigen epitopesis dinitrophenyl; among the enzyme substrates are NADH and NADPH; andamong the enzymes are horseradish peroxidase and alkaline phosphatase.

Other detectable labels useful in the invention, to mention just a few,include fluorescein isothiocyanates, dinitrophenylisothiocyanates,fluorodinitrobenzene, N-hydroxysuccinimidylbiotin,N-hydroxysuccinimidyl, dinitrobenzoate, aminobutyl ethyl isoluminolisothiocyanate, active esters of carboxyfluorescein, rhodamine, biotinadducts, dioxetanes, dioxamides, carboxyacridines, and carbohydrates.

Preferred detectable labels of the invention include biotin anddigoxigenin. Preferred methods of detecting biotin and digoxigenininclude enzyme immunoassays [EIAs] that employ alkaline phosphataseconjugated to avidin or to streptavidin, or to an antibody specific fordigoxigenin.

Applications of the invention include all of the uses for bases,nucleosides, nucleotides, oligonucleotides and polynucleotides,particularly those containing labels. For instance, the presentinvention is a useful tool in recombinant DNA and other protocolsinvolving nucleic acid hybridization techniques.

More specifically, oligonucleotides and nucleic acids containing acompound of the invention can be used as hybridization probes, capableof recognizing and specifically binding to complementary nucleic acidsequences, providing thereby a means of detecting, identifying, locatingand measuring complementary nucleic acid sequences in a biologicalsample.

Biological samples include, among a great many others, blood or bloodserum, lymph, ascites fluid, urine, microorganism or tissue culturemedium, cell extracts, or the like, derived from a biological source, ora solution containing chemically synthesized protein, or an extract orsolution prepared from such fluid from a biological source. It isfurther intended to include cells, tissue and other organic matter suchas feces, food and plants.

Molecular probes containing a compound of the invention have a varietyof applications. They can be used to detect and identify viral, fungal,bacterial and parasitic nucleic acid sequences, serving thereby to aidthe diagnosis of various related human, animal and plant diseases. Theycan be similarly used for both diagnostic and quality control purposesto detect and identify viral, fungal and bacterial contaminants in foodsand for DNA typing in forensic and paternity investigations.

Molecular probes containing the invention also can be used to identifynucleotide sequences to which proteins bind specifically. For instance,probes immobilized on solid supports can isolate and purify proteinswhich bind to sequences within the probes. Probes labeled with biotin,for example, can be bound to affinity chromatography supports, ormagnetic beads bearing covalently coupled streptavidin. Proteins whichbind to such biotin-labeled probes can be separated from a biologicalsample containing a variety of other proteins. Other methods forpurifying proteins using a solid support are well known to the skilledartisan.

An oligonucleotide containing a modified nucleotide of the invention canbe used as a primer to initiate nucleic acid synthesis at locations in aDNA or RNA molecule comprising the sequence complementary to theoligonucleotide sequence. The synthesized nucleic acid strand would haveincorporated, at its 5' terminus, the oligonucleotide primer bearing theinvention and would, therefore, be detectable by exploitation of thecharacteristics of the detectable label. Two such primers, specific fordifferent nucleotide sequences on complementary strands of dsDNA, can beused in the polymerase chain reaction (PCR) to synthesize and amplifythe amount of a nucleotide sequence. The detectable label present on theprimers will facilitate the identification of desired PCR products. PCR,combined with techniques for preparing complementary DNA (cDNA) can beused to amplify various RNAs, with oligonucleotide primers again servingboth to provide points for initiation of synthesis in the cDNA duplexflanking the desired sequence and to identify the desired product.Primers labeled with the invention may also be utilized for enzymaticnucleic acid sequencing by the dideoxy chain-termination technique.

Because the invention is derived by chemical incorporation of detectablelabels into guanine or N⁷ -alkylguanine residues in a nucleic acid, theinvention can be applied as a guanine-specific or7-alkylguanine-specific adduct to measure or quantitate the amount ofDNA present in a sample. For instance, the concentration of nucleic acidcan be measured by comparing detectable labels incorporated into theunknown nucleic acid with the concentration of detectable labelsincorporated into known amounts of nucleic acid.

Such a comparative assessment can be done using biotin where therespective concentrations are determined by an enzyme-linked assayutilizing the streptavidin-alkaline phosphatase conjugate and asubstrate yielding a soluble chromogenic or chemiluminescent signal.

The present invention is further described by reference to the followingillustrative examples.

EXAMPLE 1 Reduction Using Sodium Borohydride of Substituents in DNAFormed By Ring-methylation Followed By Base Treatment

These experiments show that the aldehyde group generated by scission ofthe C8-N⁹ bond in 7-methyl guanine is reducible and could be used forincorporating a detectable label into DNA.

DNA was methylated by treatment with DMS in a neutral buffer for 10, 20or 30 minutes, resulting in the formation in the DNA of successivelyincreasing amounts of N⁷ -methylguanine. The methylated DNA was thentreated with either sodium hydroxide or with sodium hydroxide and sodiumborohydride, breaking the C8-N⁹ bond in the methylated quaninenucleotides and generating either ring-opened N⁷ -formyl-N⁷-methylguanine nucleotides or reduced ring-opened N⁷ -formyl-N⁷-methylguanine nucleotides.

The DNAs thus treated were then exposed to tritium-labeled sodiumborohydride. As shown in FIG. 5, DNA which had not been reduced withunlabeled sodium borohydride took up tritium label and the extent oftritium uptake was directly proportional to the extent of methylation.

These experiments show clearly the generation of the reducible [formyl]group by alkylation and base treatment.

EXAMPLE 2 Biotinylation of Bacteriophage X DNA, and Use of theBiotinylated X DNA to Probe a Southern Blot

By means of the procedure set forth below, ring-opened N⁷ -formyl-N⁷-methyl guanine residues were formed in λ DNA by treatment with DMS andsodium hydroxide. Biotin was attached to the carbon 8 aldehyde of the N⁷-formyl-N⁷ -methyl guanine residues in the λ DNA by reaction withbiotin-aminocaproyl hydrazide. The biotinylated λ DNA was hybridized toa Southern blot of λ DNA digested with HindIII, and detected by EIAusing a streptavidin-alkaline phosphatase conjugate. Similar experimentswere carried out using the biotinylated λ DNA to probe slot-blots of λDNA. In addition, a sandwich assay was carried out in which biotinylatedλ DNA was captured by avidin immobilized in microtiter plate wells andthen measured by ELISA.

(1) Components of the Reaction:

1. Bacteriophage Lambda DNA (λ DNA) (20 micrograms/50 microliters;Gibco-BRL; #5250SA) Chilled to 0° C.

2. Buffered dimethylsulfate (DMS), consisting of 5 microliters ofdimethylsulfate (Eastman Kodak) per milliliter 0.5M Sodium cacodylate,pH 8.0 containing 0.0001M EDTA. Chilled to 0° C.

3. 0.4N NaOH.

4. Sodium Acetate Buffer (4.0M Sodium Acetate, pH 5.6)

5. Buffered biotin aminocaproyl hydrazide, consisting of 1 mg of biotinaminocaproyl hydrazide per milliliter of sodium acetate buffer.

6. Spin columns containing a 0.9×4 cm bed of BioGel P2 (400 mesh).

(2) Reaction Protocol

(a) Methylation and Ring Opening

The methylation reaction was started by mixing 200 microliters ofbuffered DMS with 20 micrograms (50 microliters) of λ DNA andtransferring the mixture to a 25° C. water bath. The mixture wasincubated overnight hours). 50 microliters of 0.4N NaOH were added andthe mixture was incubated at 37° C. for 2 hours to generate aldehydegroups.

(b) Coupling of Biotin Hydrazide to DNA.

50 microliters of 4M sodium acetate buffer were mixed with themethylated λ DNA to give a pH of 5.6 and 200 microliters of bufferedbiotin-aminocaproyl-hydrazide were added. The mixture was incubatedovernight at 37° C. Biotinylated λ DNA was separated from unreactedbiotin-aminocaproyl-hydrazide by centrifugation through a BioGel P2 spincolumn. Recovery of DNA was 85% (17 micrograms) in the column effluent.

(3) Results

As a test for the presence of biotin in the λ DNA probe, 300 ng ofbiotinylated λ DNA per milliliter was hybridized to a Southern blot ofHindIII fragments of λ DNA (Example 5). The biotinylated λ DNA probedetected the 6.6 Kb HindIII fragment of λ DNA at an apparentconcentration of 0.9 pg against a moderate background. Hybridization toslot-blots of serially diluted HindIII fragments of λ DNA detected 100fg of target DNA, while biotinylated λ DNA captured by immobilizedavidin in microtiter plates was detected at 0.6 pg per well.

EXAMPLE 3 Biotinylation of a Synthetic Oligodeoxynucleotide

The present invention is further illustrated by the biotinylation of asynthetic oligodeoxynucleotide. 10 μg of a 48-mer oligodeoxynucleotidecorresponding to the degenerate nucleotide sequence encoding the first16 amino acids of the amino terminus of Streptococcus sobrinusglucan-binding protein (gbpl) is diluted in 50 μl of deionized water.All subsequent reactions are carried out as described above in EXAMPLE2, using the synthetic oligonucleotide in place of λ DNA. Assaying thereaction products by electrophoresis in 20% polyacrylamide gels,Southern blotting the oligonucleotides onto a membrane, and detectingbiotinylated species in the blot with streptavidin-alkaline phosphataseconjugate showed the presence of biotin in the oligonucleotides.

EXAMPLE 4 Biotinylation of Transfer RNA

RNA can be biotinylated using methods of the invention set forthhereinabove, and detailed procedures similar to those set forth inEXAMPLE 2.

For labeling RNA, deionized water for use in preparing buffers and othersolutions is autoclaved and treated with 200 μl diethylpyrocarbonate perliter of water to inhibit the enzyme RNase. Where possible, buffers alsoare autoclaved. RNase-free, sterile plasticware is used in all labelingprocedures.

5 μl of buffered DMS are thoroughly mixed with 195 μl of 0.5Mcacodylate, 0.01M EDTA, pH 8.0 and 50 μl (500 ng) of tRNA in a 1.5 mlmicrocentrifuge tube. The mixture is incubated at 25° C., in a waterbath, overnight.

Following the 25° C. incubation, sufficient 0.4 N NaOH is added to thereaction mixture to achieve a pH of 10.0. The reaction mixture is thenincubated at 25° C. for 1 hour to open the imidazole rings of N⁷-methylguanosine residues formed in the tRNA as a result of theincubation with DMS.

The reaction is stopped by addition of 50 μl of 4.0M sodium acetatebuffer to adjust the reaction mixture to pH 5.6.

Biotinylation of the ring-opened N⁷ -formyl-N⁷ -methylguanosine residuesin the tRNA is carried out using biotin aminocaproyl hydrazide in thesame manner as described in EXAMPLE 2 for λ DNA. Separation of thebiotin-labeled tRNA from excess biotin aminocaproyl hydrazide also isaccomplished as described for biotinylation of λ DNA in Example 2, usinga gel permeation spin column. Biotin-RNA prepared in this manner may beused immediately or stored frozen at -20° C.

The presence of biotin in tRNA (and in other RNA's prepared in this way)is detected and quantitated by EIA analysis of dot or slot blots ofserial dilutions of the biotinylated RNA diluted in 10 mM Tris--HCl, 1mM EDTA, pH 8.0. The total amount of RNA is estimated by ethidiumbromide fluorescence, while the amount of biotin incorporation isestimated in the colorimetric ELISA by comparison with a standardpreparation of biotinylated RNA.

EXAMPLE 5 DNA Titration Using Biotinylated λ DNA as a HybridizationProbe

Bacteriophage λ DNA was biotinylated as described in EXAMPLE 2.Unlabeled λ DNA digested with HindIII was resolved into its fragments byelectrophoresis in a 1% agarose gel. The HindIII digested λ DNA wasapplied to the gel lanes in amounts of 150 ng (lane 1), 10 ng (lane 2),1 ng (lane 3), 500 pg (lane 5), 50 pg (lane 7), 10 pg (lane 9) and 1 pg(lane 11).

After electrophoresis the DNA was Southern blotted onto Nytran nylonmembranes and hybridized to biotinylated λ DNA following proceduresdescribed in Selden, "Analysis of DNA Sequences by Blotting andHybridization," Unit 2.9, pages 2.9.1-2.9.10 in Ausubel et al., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons 1991).

After blotting and before hybridization the membranes were prehybridizedat 42° C. for 6 hours. Denatured biotinylated λ DNA was diluted to 300ng/ml in hybridization buffer and incubated with the membranes untilhybridization was complete. The hybridized membranes were then washedand blocked by 30 minutes incubation in 3% BSA in PBS at roomtemperature. Blocked membranes were incubated 30 minutes at roomtemperature in streptavidin-alkaline phosphatase (SAAP) conjugatediluted 1:12,000 in PBS and then washed 4 times (10 minutes per wash) inPBS.

Bound SAAP was detected by incubation of membranes in 0.165 mg/ml5-bromo-4-chloro-3-indolyl phosphate ("BCIP") and 0.33 mg/ml nitro bluetetrazolium ("NBT") in 0.1M Tris pH 9.6, 50 mM MgCl₂, 100 mM NaCl. Thesmallest amount of HindIII λ DNA detected by this chromogenic stain was0.9 pg, in band 4 (6.6 Kbp) of lane 9.

Additional sensitivity was obtained using a chemiluminescent substrate.Blots containing 100 fg in lane 11 were treated as above, except thatthey were developed using a chemiluminescent substrate for alkalinephosphatase, in this case "LUMIPHOS 530" (Lumigen, Inc.). By these means49 fg (band 1 in lane 11) of λ DNA was detected, against a heavybackground, approximately 20-fold better than with the chromogenicsubstrates. cl EXAMPLE 6

Detection of the E. coli lac z Gene By Hybridization with NiotinylatedPhage M13mp19 DNA

Single-stranded M13mp19 DNA, which contains one copy of the alphafragment of the lac Z gene, was biotinylated as described for whole λDNA in EXAMPLE 2, and used to probe E. coli genomic DNA in a slot-blotfor the presence of the endogenous E. coli lac Z gene.

Thus, genomic DNA of E. coli strain Y1090 ("Y1090 DNA") wasserial-diluted, in 10-fold steps, in 1X SSC, providing concentrations ofgenomic DNA ranging from 100 ng/100 μl to 1 pg/100 μl. The dilutions ofY1090 DNA were applied to Nytran membranes, 100 μl per slot, using aslot-blot apparatus (Life Technologies Inc., Bethesda ResearchLaboratories).

Slot-blot membranes were then baked 1 hour at 80° C., prehybridized andhybridized as described in EXAMPLE 5. Biotinylated M13mp19 was dilutedto 300 ng/ml in the hybridization solution. The slot-blot was incubatedin 5 ml of hybridization solution at 42° C. overnight. Processing of theprobe slot-blots, through incubation with SAAP and development with theNBT/BCIP substrate were as described in EXAMPLE 5.

The lac Z gene was detected in slots containing 100 pg to 1 μg of E.coli Y1090 genomic DNA. Detection of lac Z in 100 pg genomic DNArepresents visualization of about 75 μg of lac Z gene, since a singlecopy of the 3.51×10³ bp lac Z gene is present in the E. coli genome,which contains 4.7×10⁶ bp, i.e., (3.5×10³/4.7×10⁶)(100×10⁻¹²)=7.45×10⁻¹⁴.

EXAMPLE 7 Use of Biotinylated Oligonucleotide as a Hybridization Probefor Southern Blots of Restricted Genomic DNA.

The gbpl oligonucleotide biotinylated in EXAMPLE 3 serves as ahybridization probe to detect the gbpl gene in a Southern blot of EcoRIrestricted Streptococcus sobrinus 6715-49 genomic DNA. Streptococcalgenomic DNA is digested to completion with EcoRI, then 10 μg aliquotsare resolved in multiple lanes by electrophoresis overnight in a 1%agarose gel in 1 X Tris-Acetate-EDTA buffer ("TAE," 20 X TAE buffercontains 242 g Tris base, 57.1 ml glacial acetic acid, 37.2 g EDTA at pH8.5). A control lane contains 10 μg of E. coli Y1090 genomic DNAdigested to completion with EcoRI.

The Southern blot is prepared, prehybridized and hybridized by theprocedures described in EXAMPLE 5. The biotinylated gbploligonucleotidehybridization probe is diluted to a final concentrationof 300 ng/ml in hybridization buffer. After hybridization overnight at42° C. in 5 ml per filter of hybridization solution, excess probe iswashed away and the annealed biotinylated. gbpl oligonucleotide probe isvisualized as described in EXAMPLE 5. A single band of target DNA, ofapproximately 8 kbp, is detected in all lanes containing S. sobrinus DNAbut not in the E. coli Y1090 DNA control lane. This result replicates asimilar experiment utilizing this oligonucleotide, 5'-end labeled with³² p, as a hybridization probe.

EXAMPLE 8 T_(m) of Biotinylated λ DNA.

λDNA was biotinylated in the manner described in EXAMPLE 2. Meltingtemperatures (T_(m)) were determined for biotinylated λ DNA andunmodified λ DNA. Each DNA was dissolved at 15 μl/ml in 1X SSC buffer(150 mM NaCl, 15 mM sodium citrate, pH 7.0). DNA absorbance wasmonitored at 260 nm while increasing the temperature in increments of 5°C., from room temperature to 100° C. The T_(m) of biotinylated λDNA was84° C., while that of unmodified λDNA was 89° C.

EXAMPLE 9 Biotinylation of dGTP and End-Labeling DNA Using theBiotinyl-dGTP and Terminal Deoxynucleotidyl Transferase

Deoxyguanosine triphosphate (dGTP), dissolved at 1 mg/ml in cacodylatebuffer is biotinylated as described in EXAMPLE 2, with the omission ofspin-column chromatography. The biotin-labeled dGTP ("Bio-dGTP") isseparated from unbound biotin aminocaproyl hydrazide by reverse-phasechromatography on a C18 column, with a 0 to 80% gradient of acetonitrile(CH₃ CN). Bio-dGTP is freed from acetonitrile in a rotary evaporatorunder vacuum at 37° C., then redissolved in deionized water.

To biotinylate the S. sobrinus gbpl 48-mer oligonucleotide usingterminal deoxynucleotidyl transferase (TdT), the reaction mixturecontains:

100 mM sodium cacodylate, pH 7.0

1 mM CoCl₂

0.1 mM dithiothreitol

4 pmoles oligonucleotide (as 3' termini)

20 μm Bio-dGTP

2.5 μg bovine serum albumin

10 International Units terminal deoxynucleotidyl transferase

The reaction is incubated at 37° C. for 30 minutes and terminated byadding 2 μl of 0.5M EDTA. End-labeled oligonucleotide is separated fromunincorporated Bio-dGTP by centrifugation through a BioGel P2 spincolumn. If desired, TdT is removed by phenol extraction and theoligonucleotide is precipitated from the aqueous phase by the additionof two volumes of absolute ethanol and incubation at -70° C. for atleast 1 hour. The use of CoCl₂ allows end-labeling of ribonucleotides aswell as deoxyribonucleotides.

EXAMPLE 10 A Competitive Enzyme Immunoassay for DNA

Microtiter plate wells ("IMMULON 2" microtiter plates from DynatechLaboratories, Inc.) were coated by overnight incubation at roomtemperature with 300 μl/well of 5μg/ml avidin in PBS (Pierce ChemicalCompany), and then blocked 30 minutes with 300 μl/well of 0.1M NH₄ Cl.The reporter enzyme for the assay was biotinylated alkaline phosphatase("BAP"; Pierce Chemical Company) diluted from a 1 mg/ml PBS stocksolution. Useful dilutions of BAP for the detection of nanogramquantities of DNA ranged from 1/40,000 through 1/100,000, whiledetection of picogram levels of DNA was optimum at BAP dilutions of1/100,000 through 1/140,000 (see insets in FIGS. 6 and 7).

E. coli Y1090 genomic DNA was biotinylated by the procedure of EXAMPLE2. Biotinylated DNA was diluted in PBS to provide two dilution series: 1to 100 of biotinylated DNA per 200μl and 1 to 100pg of biotinylated DNAper 200 μl. 200 μl aliquots of each biotinylated DNA dilution series, orof PBS only, were incubated in avidin-coated wells for two hours at roomtemperature on a rotary shaker. Following three washes with PBS, thewells received 200 μl aliquots of BAP at appropriate dilutions andincubation was continued, as above, for two more hours.

Plates were washed six times with PBS and then 200 μl aliquots ofp-nitrophenylphosphate (4 mg/ml 10% diethanolamine, 0.5 mM MgCl₂, pH9.8) were added to each well. Rates of the ensuing enzyme reactions weredetermined in a kinetic platereader (Easy Reader EA 400 AT; SLTLabinstruments) at 405 nm. End-point optical density values at 405 nmwere determined after 1 hour incubation at room temperature.

FIGS. 6 and 7 show values of percent inhibition for the nanogram andpicogram ranges, respectively. Insets in these figures compare responsesat various reporter enzyme dilutions. These competitive assays arecapable of measuring DNA over concentration ranges of 1 to 25 ng/200 μl(FIG. 6) and 1 to 100 pg/200 μl (FIG. 7).

What is claimed is:
 1. A compound according to the following formula:##STR4## wherein, A is --H or --OH;B is --H, --OH, mono-, di- ortriphosphate, or a protecting group for chemical polynucleotidesynthesis; D is --H, --OH, mono-, di- or triphosphate, or a protectinggroup for chemical polynucleotide synthesis; E is an alkyl group; F is abond or linking moiety, and; G is a detectable label or a blockinggroup.
 2. A compound according to claim 1, wherein E is methyl, ethyl orbenzyl, and G is an affinity moiety or a hapten.
 3. A compound accordingto claim 1, wherein:A is --H or --OH; B is --H, --OH, or mono-, di- ortriphosphate; D is --H, --OH, or mono-, di- or triphosphate; E is analkyl group; F is a bond or linking moiety, and; G is a detectable labelstable to an enzymatic polynucleotide labeling reaction or a blockinggroup for an enzymatic labeling reaction.
 4. A compound according toclaim 1, wherein:A is --H or --OH; B is a phosphoramidite or otherprotecting group for polynucleotide synthesis; D is a trityl or otherprotecting group for polynucleotide synthesis; E is an alkyl group; F isa bond or linking moiety, and; G is a detectable label stable topolynucleotide synthesis or a blocking group for polynucleotidesynthesis.
 5. A polynucleotide comprising a compound according to thefollowing formula: ##STR5## wherein: A is --H or --OH;B is --H, --OH, ormono-, di- or triphosphate, or a phosphodiester bond; D is --H, --OH ormono-, di- or triphosphate, or a phosphodiester bond; E is an alkylgroup; F is a bond or linking moiety, and; G is a detectable label.
 6. Apolynucleotide according to claim 5, which is an oligonucleotide primerfor elongation.
 7. An oligonucleotide according to claim 6, comprising afirst sequence that hybridizes to a target DNA and a sequence 5' to saidfirst sequence that encodes one or more cis-acting or trans-actingfunctions.